Apparatus and methods for investigating the binding of analytes in solution to a receptor are known. Recently, surface sensitive measuring techniques using so-called label-free techniques have been developed for measuring and quantifying biomolecular interactions. In these techniques, a receptor capable of binding to an analyte of interest is immobilised to a sensor surface, and binding of the analyte to the receptor is detected as a resulting change of a property of the sensor surface.
One type of such apparatus (with associated computer control and data-processing means), including the commercial instruments BIAcore and BIAlite (BIAcore and BIAlite are trademarks of Pharmacia Biosensor AB, Uppsala, Sweden; BIA stands for biospecific interaction analysis) has been devised, which uses the phenomenon of surface plasmon resonance (SPR) to study the binding of analytes to receptors immobilized on a sensor chip. The apparatus and theoretical background are fully described in the literature (see e.g. Jonsson, U., et al., BioTechniques 11: 620-627 (1991)). Essentially, the technique involves the immobilisation of a receptor to the special surface of a sensor chip, contacting the sensor chip with a flow of sample containing the analyte of interest, and then measuring the change in the surface optical characteristics of the sensor chip arising form the binding of interest.
With such instrumentation, for example, affinity and kinetic analysis of interactions between soluble analytes and their immobilised binding partners may readily be performed. However, in many cases, it would also be interesting to know the true solution affinity and kinetics of the interaction between two species interacting in solution. So far, such analyses have not been done with the above described type of apparatus.
Friguet, B., et al., Anal. Biochem. 210, 344-350 (1993) discloses the determination of the true affinity constant of a monoclonal antibody for its antigen. Aliquots of radiolabeled antigen at a constant concentration are incubated with the monoclonal antibody at various known concentrations of the antibody in large excess over the antigen. When equilibrium has been reached, the concentration of free antigen is determined by the binding to dextrane beads to which the same monoclonal antibody has been covalently attached. However, this approach only gives information on the equilibrium constant and no kinetic information is provided.